Anti-aging and anti-inflammatory agents

Abstract

The objective of this invention is to provide a novel active ingredient that can suppress aging-related inflammation. [Solution] Pyridoxines have the effect of suppressing the production of SASP factors, and are therefore useful as active ingredients in anti-aging and anti-inflammatory agents.

Description

Technical Field

[0001] The present invention relates to an anti-aging inflammation inhibitor.

Background Art

[0002] Most normal somatic cells have a determined number of divisions and cannot divide further once a certain number of divisions is reached. This state where division reaches its limit is called cellular senescence, and cells that have divided up to the limit number of divisions are called senescent cells. It has been found that senescent cells cause chronic inflammation through the secretion of inflammatory cytokines via the senescence-associated secretory phenotype (SASP).

[0003] Known SASP factors include inflammatory cytokines (interferon (IL)-1α / β, IL-6, IL-8, TNF-α), chemokines (CCL, CXCL), extracellular matrix degrading enzymes (MMPs), growth factors (EGF, VEGF, PGDF), extracellular matrix (fibronectin, collagen), etc. (Non-Patent Document 1).

[0004] CCL2, which is an example of a C-C motif chemokine receptor (CCL), is an important regulatory factor in periodontal disease and a therapeutic target. Specifically, it has been reported that administration of bindarit, a CCL2 inhibitor, to a periodontal disease model was effective in reducing osteoclasts (Non-Patent Document 2).

[0005] Furthermore, it has been reported that the expression localization of CCL2 changes during the progression of periodontal disease. Specifically, the localization of CCL2 transitions in the order of (1) macrophages on the alveolar bone surface, (2) macrophages (the number of CCL2-positive macrophages further increases), immune cells infiltrating the periodontal tissue, intravascular cells, and gingival cells, (3) further to the periodontal ligament, and (4) the alveolar bone surface and root surface (the number of CCL2-positive periodontal ligament cells decreases) (Non-Patent Document 3).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] While some compounds useful as SASP factor inhibitors, such as CCL2 inhibitors, are already in practical use as pharmaceuticals, they require a doctor's prescription and careful handling. On the other hand, considering that periodontal disease and other conditions caused by SASP in senescent cells in periodontal tissue are common, it is desirable to be able to select a more versatile active ingredient that can suppress the production of SASP factors.

[0008] Therefore, the purpose of this disclosure is to provide a new active ingredient that can suppress aging inflammation. [Means for solving the problem]

[0009] The inventors of this invention conducted diligent research to solve the aforementioned problems and discovered that pyridoxines have the effect of suppressing the production of SASP factors CCL2 and MMP-1. The present invention was completed by further research based on this finding.

[0010] In other words, this disclosure provides inventions in the following embodiments. Item 1. Anti-aging and anti-inflammatory agents containing pyridoxines. Item 2. An anti-aging inflammation agent as described in Item 1, used to suppress aging inflammation in periodontal tissue. Item 3. An anti-aging and anti-inflammatory agent according to item 1 or 2, used as an oral composition. Item 4. An anti-aging and anti-inflammatory agent according to any one of items 1 to 3, for use in application to CCL2 expression sites in periodontal tissue. Item 5. An anti-aging and anti-inflammatory agent described in any of items 1 to 4, used for the prevention or improvement of periodontal disease. Item 6. A method for suppressing aging inflammation, comprising administering an effective amount of pyridoxine to subjects requiring suppression of aging inflammation. Item 7. Use of pyridoxines for the manufacture of compositions for suppressing aging and inflammation. Item 8. Pyridoxines for use in the prevention or treatment of diseases associated with aging inflammation. Item 9. Non-therapeutic use of pyridoxines to suppress aging inflammation. [Effects of the Invention]

[0011] According to this disclosure, age-related inflammation can be suppressed by using pyridoxines as a novel active ingredient. [Brief explanation of the drawing]

[0012] [Figure 1] This disclosure demonstrates the inhibitory effect of the anti-aging and anti-inflammatory agent (pyridoxine hydrochloride) on the production of SASP factor (CCL2) in senescent cells. [Figure 2] This study demonstrates the effect of a common anti-inflammatory component (glycyrrhetinic acid) on SASP factor (CCL2) production in senescent cells. [Figure 3] This study demonstrates the effect of a common anti-inflammatory component (allantoin) on SASP factor (CCL2) production in senescent cells. [Figure 4] This study shows the effect of a common anti-inflammatory component (alcloxa) on SASP factor (CCL2) production in senescent cells. [Figure 5] This study demonstrates the effect of a common anti-inflammatory component (ε-aminocaproic acid) on SASP factor (CCL2) production in senescent cells. [Modes for carrying out the invention]

[0013] Hereinafter, the anti-aging inflammation inhibitor of the present disclosure will be described in detail. In this specification, a numerical range indicated by two numerical values and "~" shall include the two numerical values as the lower limit value and the upper limit value. For example, the notation of 2~15% by weight means 2% by weight or more and 15% by weight or less.

[0014] Active ingredients The anti-aging inflammation inhibitor of the present disclosure contains pyridoxines as an active ingredient. The pyridoxines used in the present disclosure are pyridoxine, pyridoxal, pyridoxamine, and their salts.

[0015] When pyridoxines are in the form of salts, the types of the salts are not particularly limited as long as they are pharmaceutically or physiologically acceptable. Examples include inorganic acid salts. More specifically, hydrochloride, sulfate, nitrate, hydrobromide, phosphate, etc. are included.

[0016] These pyridoxines may be used alone or in combination of two or more.

[0017] Among these pyridoxines, from the viewpoint of enhancing the anti-aging inflammation effect, preferably pyridoxine or its salt, more preferably pyridoxine salt, and still more preferably pyridoxine hydrochloride are included.

[0018] In the anti-aging inflammation inhibitor of the present disclosure, the content of pyridoxines is not particularly limited and may be appropriately set according to the type of pyridoxines used, the degree to which the anti-aging inflammation effect should be imparted, and the dosage form and use of the anti-aging inflammation inhibitor. Examples include 0.001~10% by weight. From the viewpoint of enhancing the anti-aging inflammation effect, the content of pyridoxines is preferably 0.007~10% by weight, more preferably 0.0015~10% by weight, still more preferably 0.02~10% by weight, 0.03~10% by weight, even more preferably 0.04~10% by weight, 0.04~5% by weight, 0.04~1% by weight, 0.04~0.5% by weight, or 0.04~0.1% by weight.

[0019] Other ingredients The anti-aging and anti-inflammatory agents disclosed herein may or may not contain other pharmacological components in addition to the active ingredients described above, as needed. Examples of such pharmacological components, whether present or absent, include antihistamines, local anesthetics (dibucaine hydrochloride, ethyl aminobenzoate, etc.), bactericides (isopropylmethylphenol, cetylpyridinium chloride, benzethonium chloride, chlorhexidine hydrochloride, chlorhexidine gluconate hydrochloride, triclosan, lauroyl sarcosine salt, hinokitiol, etc.), astringents (sodium chloride, Phellodendron amurense extract, Angelica acutiloba extract, Paeonia lactiflora extract, allantoin, alcloxa, etc.). Other examples include anti-inflammatory agents (sodium azulene sulfonate, tranexamic acid, dipotassium glycyrrhizate, β-glycyrrhetinic acid, ε-aminocaproic acid, lysozyme chloride, methyl salicylate, cineole, etc.), antioxidants (pycnogenol, ascorbic acid, ascorbic acid glucoside), cell activators (allantoin, etc.), skin protectants, blood circulation promoting ingredients (tocopherol acetate, tocopherol nicotinate, panthenol, etc.), hemostatic agents (carbazochrome, etc.), and mucopolysaccharides.

[0020] Furthermore, the anti-aging and anti-inflammatory agents of this disclosure may or may not contain base materials and / or additives other than the components described above, as necessary, in order to obtain the desired formulation form. Such base materials and additives, whether present or absent, are not particularly limited to the extent that they are pharmaceutically or physiologically acceptable, but examples include water, lower (1-5 carbon) monohydric alcohols, polyhydric alcohols, natural oils and fats, hydrocarbon oils, ester oils, fatty acid alkyl esters, fatty acids, fatty acid esters, higher (6 or more carbon) monohydric alcohols, cholesterol, glyceryl tri-2-ethylhexanoate, cetyl 2-ethylhexanoate, silicone oil; emulsifiers; cleansing components (surfactants, sodium bicarbonate, etc.), cooling agents, preservatives, fragrances, colorants, viscosity modifiers, pH adjusters, wetting agents, abrasives, stabilizers, antioxidants, UV absorbers, chelating agents, adhesives, buffers, solubilizers, preservatives, etc.

[0021] Dosage form, formulation, and product classification The dosage form of the anti-aging inflammation agent of this disclosure is not particularly limited and may be liquid, semi-solid, solid, or any other form. The anti-aging inflammation agent of this disclosure may be a non-emulsified composition or an emulsified composition, but a non-emulsified composition is preferred, and a non-emulsified aqueous composition is preferred.

[0022] The anti-aging and anti-inflammatory agents disclosed herein can be formulated in the form of gels, creams, lotions, emulsions, liquids, ointments, pastes, jellies, aerosols, powders, granules, capsules, candies, drops, lozenges, tablets, chewables, gummies, edible films, etc. These formulations can be prepared by compounding them using additives appropriate to the formulation, in accordance with known methods described in the General Provisions for Formulations of the Seventeenth Revised Japanese Pharmacopoeia, etc.

[0023] Furthermore, product classifications of anti-aging and anti-inflammatory agents in this disclosure include topical preparations and oral preparations, more specifically, topical pharmaceuticals, topical quasi-drugs, oral pharmaceuticals, and oral quasi-drugs. Preferably, product classifications of anti-aging and anti-inflammatory agents in this disclosure include mucosal preparations. Mucosal preparations include oral preparations and other mucosal preparations (e.g., suppositories and vaginal preparations), with oral preparations being preferred. Specifically, these include mouthwash, gargles, dental creams, oral ointments, toothpastes (paste, gel toothpaste, liquid toothpaste (dental rinse)), oral tablets (buccal tablets, gums, lozenges, adhesive tablets, etc.), oral capsules (for oral spray), oral sprays, etc.

[0024] Purpose The anti-inflammatory agents of this disclosure are used to suppress inflammation caused by cells that have divided to their limit, i.e., senescent cells. Preferably, the anti-inflammatory agents of this disclosure are used to suppress inflammation based on CC motif chemokine receptor 2 (CCL2).

[0025] The anti-aging inflammation inhibitors of this disclosure are preferably used to suppress anti-aging inflammation in the oral cavity, more preferably in periodontal tissue, and even more preferably applied to CCL2 expression sites in periodontal tissue.

[0026] The anti-aging and anti-inflammatory agents of this disclosure are preferably used for the prevention or improvement of periodontal disease. The periodontal disease may be either gingivitis or periodontitis. The stage of periodontal disease is not particularly limited and may be stage I, stage II, stage III, or stage IV. The grade of periodontal disease is also not particularly limited and may be grade A, grade B, or grade C. The progression stage of periodontal disease based on the localization of CCL2 is also not particularly limited and may be any of the following: (1) a stage in which CCL2 is localized in giant cells on the surface of the alveolar bone; (2) a stage in which the number of CCL2-positive giant cells increases compared to stage (1), and CCL2 is also localized in immune cells, endovascular cells, and gingival cells infiltrating the periodontal tissue; (3) a stage in which CCL2 is also localized in the periodontal ligament; or (4) a stage in which the number of CCL2-positive periodontal ligament cells decreases compared to stage (3), and CCL2 is localized on the surface of the alveolar bone and the root surface. If the anti-aging and anti-inflammatory agent of this disclosure is a topical agent, it may be applied at either the prevention stage or the progression stage of periodontal disease, but it is preferable to apply it at the stage described in (2) and / or (3) above, where CCL2 is present at a relatively shallow location from the surface of the periodontal tissue (including the inner surface of the periodontal pocket).

[0027] The anti-aging and anti-inflammatory agents of this disclosure can preferably also be used to suppress the production of MMP-1 (matrix metalloproteinase-1). More specifically, the anti-aging and anti-inflammatory agents of this disclosure can protect periodontal tissue from MMP-1-induced destruction by suppressing MMP-1 production in the periodontal ligament, and can therefore be used to suppress gingival tissue destruction, and consequently to suppress or improve gingival recession.

[0028] Since senescent cells exist regardless of age or sex, inflammation caused by senescent cells, i.e., senescent inflammation, can also occur regardless of age or sex. Therefore, the target population for the senescent inflammation inhibitors disclosed herein is not particularly limited in terms of age or sex. When the senescent inflammation inhibitors disclosed herein are used for the prevention or improvement of periodontal disease, target populations include men or women in late infancy, childhood, adolescence, adulthood, middle age, or old age, and among these, men or women in middle age or old age with a high accumulation of senescent cells are preferred. When the senescent inflammation inhibitors disclosed herein are used for the prevention or improvement of periodontal disease, target populations among women further include women in pregnancy, childbirth, ovulation, luteal phase, menopause, and post-menopausal periods, as well as women taking hormonal drugs such as oral contraceptives. [Examples]

[0029] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0030] Test Example 1 (1) Test method 1. Cellular senescence was induced in cultured human gingival fibroblasts (HGnF cells) by treating them with Doxorubicin (100 ng / mL) for 13-14 days to produce senescent HGnF cells (hereinafter referred to as "senescent cells"). For each test substance, cells with the same viability and proliferation rate were used as senescent cells. Furthermore, cells that showed CCL2 expression more than twice the amount of non-senescent cells (described later) after senescence induction were selected as senescent cells within a reasonably reproducible range. 2. Separately, HGnF cells that had not undergone senescence induction (hereinafter referred to as "non-senescent cells") were prepared. 3. Senescent cells were cultured for 24 hours after being added to the test substances: pyridoxine hydrochloride, glycyrrhetinic acid, allantoin, alcloxa, or ε-aminocaproic acid, at the concentrations shown in Figures 1 to 7. In this test system, it was confirmed that the test substances were non-cytotoxic at all tested concentrations. 4. After culturing, RNA was extracted from all cells using the Relia Prep RNA Cell Miniprep System (Promega) according to the product protocol. 5. After extraction, 1 μl of Random Primer (Takara Bio) was added to 1 μg of RNA, and the sample was adjusted to 12 μl with RN-ase Free Water. The sample was then heat-treated at 65°C for 5 minutes using a T-Personal Thermocyler (Biometra) to denature the RNA, and then rapidly cooled on ice. 6. Add the reverse transcription reaction mixture (4 μl of 5×RT Buffer, 2 μl of 10 mM dNTPs, 1 μl of RNase Inhibitor, 0.5 μl of ReverTraAce, 0.5 μl of RNase-Free H2O (all reagents except H2O are from TOYOBO)) to the RNA-denatured sample, and adjust to a final volume of 20 μl. The resulting sample was reverse transcribed to cDNA using a T-Personal Thermocyler (conditions: 30°C for 10 minutes → 42°C for 60 minutes → 85°C for 5 minutes → 4°C Hold). 7. Quantitative real-time PCR was performed using the SYBR Green Kit (Qiagen) and CCL2-specific primers in a QuantStudio (Thermofisher Scientific) system. 8. The relative expression levels of the CCL2 gene were determined by a comparative quantitative method (ΔΔCt method). The same procedure as above was followed for non-senescent or senescent cells, except that the test substance was not added.

[0031] (2) Results As shown in Figure 1, aging gingival fibroblasts (without pyridoxine hydrochloride addition) showed a significantly increased expression level of CCL2 compared to non-aging gingival fibroblasts. On the other hand, when pyridoxine hydrochloride was added to aging gingival fibroblasts, CCL2 expression decreased in a concentration-dependent manner. In other words, it was found that pyridoxine hydrochloride can suppress aging inflammation in gingival fibroblasts by inhibiting the production of CCL2, a SASP factor in aging gingival fibroblasts.

[0032] On the other hand, as shown in Figures 2 to 7, glycyrrhetinic acid, allantoin, alcloxa, and ε-aminocaproic acid, which are common anti-inflammatory components used in oral compositions, were all unable to suppress the production of CCL2, a SASP factor in aging gingival fibroblasts; in other words, no inhibitory effect was observed against aging inflammation of gingival fibroblasts.

[0033] Test Example 2 (1) Test method 1. 75cm in advance 2 Non-senescent periodontal ligament fibroblasts "nSNCs" (ScienCell Research Laboratories) cultured in cell culture flasks, or periodontal ligament fibroblasts "SNCs" in which cellular senescence was induced by culturing nSNCs in the presence of 100 ng / mL doxorubicin for 19 days, were used in a 1.5 × 10⁶ sample. 5 Seeds were seeded into a 6-well plate to achieve a cell / well ratio and incubated overnight (37°C, 5% CO2). 2. After culturing, the test substance (pyridoxine hydrochloride; "VB6") was added at the specified concentrations listed in Table 1, and the cells were incubated for 24 hours (37°C, 5% CO2). In this test system, it was confirmed that the test substance was non-cytotoxic at the tested concentrations. 3. From the cultured cells, ReliaPrep TM Total RNA was extracted using RNA Miniprep Systems (Promega). 4. After extraction, 1 μl of Random Primer (Takara Bio) was added to 1 μg of RNA, and the sample was adjusted to 12 μl with RN-ase Free Water. The sample was then heat-treated at 65°C for 5 minutes using a T-Personal Thermocyler (Biometra) to denature the RNA, and then rapidly cooled on ice. 5. Add the reverse transcription reaction mixture (4 μl of 5×RT Buffer, 2 μl of 10 mM dNTPs, 1 μl of RNase Inhibitor, 0.5 μl of ReverTraAce, 0.5 μl of RNase-Free H2O (all reagents except H2O are from TOYOBO)) to the RNA-denatured sample, and adjust to a final volume of 20 μl. The resulting sample was reverse transcribed to cDNA using a T-Personal Thermocyler (conditions: 30°C for 10 minutes → 42°C for 60 minutes → 85°C for 5 minutes → 4°C Hold). 6. Quantitative real-time PCR was performed using the SYBR Green Kit (Qiagen) and primers specific to the SASP gene (MMP-1) at QuantStudio (Thermofisher Scientific). 7. The relative expression levels of each gene were determined by comparative quantitative analysis (ΔΔCt method). The procedure was the same as above, except that the test substance was not added to non-senescent cell nSNCs or senescent cell SNCs, and the relative expression levels of the SASP gene were determined. The test was performed four times, and the average value of the relative expression levels of the SASP gene was derived.

[0034] (2) Results The results are shown in Table 1. As shown in Table 1, the expression level of MMP-1, a SASP factor, was significantly increased in aged periodontal ligament fibroblasts (SNCs) compared to non-aging periodontal ligament fibroblasts (nSNCs). On the other hand, when pyridoxine hydrochloride VB6 was added to aged periodontal ligament fibroblasts (SNCs), the expression level of the SASP factor decreased in a concentration-dependent manner. In other words, it was found that pyridoxine hydrochloride can suppress aging inflammation in periodontal ligament fibroblasts by suppressing the production of MMP-1, a SASP factor, in periodontal ligament fibroblasts.

[0035] [Table 1]

Claims

1. An anti-aging and anti-inflammatory agent containing pyridoxines.

2. An anti-aging inflammation agent according to claim 1, used to suppress aging inflammation in periodontal tissue.

3. An anti-aging and anti-inflammatory agent according to claim 1, used as an oral composition.

4. An anti-aging and anti-inflammatory agent according to claim 1, for use in application to CCL2 expression sites in periodontal tissue.

5. An anti-aging inflammation agent according to claim 1, used for the prevention or improvement of periodontal disease.

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